Ras is the name given to a family of related proteins found inside cells, including human cells. All Ras protein family members belong to a class of protein called small GTPase, and are involved in transmitting signals within cells (cellular signal transduction). All Ras proteins are related in 3D structure and regulate diverse cell behaviors.
When Ras is ‘switched on’ by incoming signals, it subsequently switches on other proteins, which turn on genes involved in cell growth, differentiation and survival. As a result, mutations in ras genes can lead to the production of permanently activated Ras proteins. This can cause inappropriate and overactive signaling inside the cell, even in the absence of incoming signals, which may permanently turn on genes involved in cell growth, differentiation and survival. As a result, mutations in ras genes can lead to the production of permanently activated Ras proteins.
Overactive Ras signaling can ultimately lead to cancer. Ras is the most common oncogene in human cancer—mutations that permanently activate Ras are found in 15% of all human tumors and up to 90% in certain types of cancer (e.g. pancreatic cancer). The clinically most notable members of the Ras subfamily are H-Ras, N-Ras and K-Ras, mainly for being implicated in many types of cancer. Inappropriate activation of the gene has been shown to play a key role in signal transduction, proliferation and malignant transformation.
Ras GTPases operate as molecular switches in signaling pathways that control cell proliferation. Ras normally oscillates between an active, GTP-bound and inactive GDP-bound state in a cycle that is tightly controlled by guanine nucleotide exchange factors and GTPase activating proteins. Oncogenic Ras mutations occur in ˜15% of all human tumors and directly contribute to malignant transformation by locking Ras in the GTP-bound state leading to constitutive activation of downstream signaling pathways. For example, oncogenic K-ras mutations occur in 90% of pancreatic, 45% of colorectal and 35% of lung carcinomas (Bos J L. ras oncogenes in human cancer: a review. Cancer Res. 1989; 49 (17):4682-9 and Downward J. Targeting RAS signalling pathways in cancer therapy. Nat Rev Cancer. 2003; 3 (1):11-22). Many small molecule inhibitors of kinases downstream of K-ras have been developed as anticancer therapeutics. With the Raf-MAPK cascade, potent inhibitors of BRaf, CRaf and MEK are in clinical use (Ribas A and Flaherty K T. BRAF targeted therapy changes the treatment paradigm in melanoma. Nat Rev Clin Oncol. 2011; 8(7): 426-33; Kefford H A R, Brown M P, Millward M, Infante J R, Long G V, Ouellet D, Curtis M, Lebowitz P F, Falchook G S. Phase I/II study of GSK2118436, a selective inhibitor of oncogenic mutant BRAF kinase, in patients with metastatic melanoma and other solid tumors. J. Clin. Oncol. (Meeting Abstracts) 2010; 28, 8503; Poulikakos P I and Rosen N. Mutant BRAF melanomas—dependence and resistance. Cancer Cell. 2011; 19(1): 11-5; Joseph E W, Pratilas C A, Poulikakos P I, Tadi M, Wang W, Taylor B S, Halilovic E, Persaud Y, Xing F, Viale A, Tsai J, Chapman P B, Bollag G, Solit D B and Rosen N. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc Natl Acad Sci USA. 2010; 107(33):14903-8; Flaherty K T, Puzanov I, Kim K B, Ribas A, McArthur G A, Sosman J A, O'Dwyer P J, Lee R J, Grippo J F, Nolop K and Chapman P B. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010; 363(9):809-19; Infante J R, Fecher L A, Nallapareddy S, Gordon M S, Flaherty K T, Cox D S, DeMarini D J, Morris S R, Burris H A and Messersmith W A. Safety and efficacy results from the first-in-human study of the oral MEK 1/2 inhibitor GSK1120212. J. Clin. Oncol. (Meeting Abstracts) 2010; 28, 2503; Poulikakos P I and Solit D B. Resistance to MEK inhibitors: should we co-target upstream? Sci. Signal. 2011; 4 (166), pe16; Corcoran R B, Settleman J, and Engelman J A. Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF mutant cancers. Oncotarget. 2011; 2(4): 336-346; Halilovic E and Solit D B. Therapeutic strategies for inhibiting oncogenic BRAF signaling. Curr Opin Pharmacol. 2008; 8(4):419-26; Greger J G, Eastman S D, Zhang V, Bleam M R, Hughes A M, Smitheman K N, Dickerson S H, Laquerre S G, Liu L, and Gilmer T M. Combinations of BRAF, MEK, and PI3K/mTOR Inhibitors Overcome Acquired Resistance to the BRAF Inhibitor GSK2118436 Dabrafenib, Mediated by NRAS or MEK Mutations. Mol Cancer Ther. 2012; 11 (4):909-20).
However the clinical responses to Raf inhibitors can be relatively short-lived, with treatment failure and tumor progression occurring due to acquired resistance, primarily as a result of secondary mutations in the oncogenic BRaf or other proteins such as N-ras or MEK (Whittaker S, Kirk R, Hayward R, Zambon A, Viros A, Cantarino N, Affolter A, Nourry A, Niculescu-Duvaz D, Springer C, and Marais R. Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci Transl Med. 2010; 2(35):35 ra41; Nazarian R, Shi H, Wang Q, Kong X, Koya R C, Lee H, Chen Z, Lee M K, Attar N, Sazegar H, Chodon T, Nelson S F, McArthur G, Sosman J A, Ribas A, and Lo R S. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. 2010; 468(7326):973-7; Wagle N, Emery C, Berger M F, Davis M J, Sawyer A, Pochanard P, Kehoe S M, Johannessen C M, Macconaill L E, Hahn W C, Meyerson M, and Garraway L A. Dissecting Therapeutic Resistance to RAF Inhibition in Melanoma by Tumor Genomic Profiling. J Clin Oncol. 2011; 29(22): 3085-96). In addition, while BRaf inhibitors inhibit the activation of BRaf/MEK/ERK in BRaf mutant cell lines, they paradoxically activate MEK/ERK signaling in cell lines with Ras mutations (Heidorn S J, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I, Dhomen N, Hussain J, Reis-Filho J S, Springer C J, Pritchard C, and Marais R. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell. 2010; 140 (2):209-21; Hatzivassiliou G, Song K, Yen I, Brandhuber B J, Anderson D J, Alvarado R, Ludlam M J, Stokoe D, Gloor S L, Vigers G, Morales T, Aliagas I, Liu B, Sideris S, Hoeflich K P, Jaiswal B S, Seshagiri S, Koeppen H, Belvin M, Friedman L S, and Malek S. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature. 2010; 464 (7287):431-5; Poulikakos P I, Zhang C, Bollag G, Shokat K M, and Rosen N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature. 2010; 464 (7287):427-30).
Approaches to directly target the Ras protein have been unsuccessful. Ras proteins undergo post translational processing to generate a lipid modified C-terminus. The cysteine residue in the C-terminal CAAX motif of the cytosolic precursors of all three Ras isoforms is isoprenylated by farnesyl protein transferase, which facilitates endoplasmic reticulum (ER) association where the AAX motif is cleaved off by Ras-converting enzyme 1 (Rce1) followed by carboxymethylation of the farnesylated cysteine by isoprenyl cysteine transferase (Icmt) (Omerovic J, Laude A J, and Prior I A. Ras proteins: paradigms for compartmentalised and isoform-specific signalling. Cell Mol Life Sci. 2007; 64(19-20):2575-89). The weak membrane binding of these proteins rendered by the farnesyl moiety is further enhanced by secondary motifs or modifications, which is the basic hexalysine patch in K-ras, and one two palmitoyl groups on cysteines 181 and 184 in N- and H-ras, respectively (Apolloni A, Prior I A, Lindsay M, Parton R G, and Hancock J F. H-ras but not K-ras traffics to the plasma membrane through the exocytic pathway. Mol Cell Biol. 2000; 20(7):2475-87; Choy E, Chiu V K, Silletti J, Feoktistov M, Morimoto T, Michaelson D, Ivanov I E, Philips M R. Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell. 1999; 98(1):69-80; Hancock J F, Paterson H, and Marshall C J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990; 63(1):133-9).
These second signal motifs also specify the trafficking routes: H- and N-ras traffic via the conventional secretory pathway, whilst preliminary data from yeast indicates that K-ras transits via a poorly understood Golgi-independent route that requires mitochondrial function and class C vps proteins (Apolloni, et al., 2000, ibid, Choy E, Chiu V K, Silletti J, Feoktistov M, Morimoto T, Michaelson D, Ivanov I E, and Philips M R. Endomembrane trafficking of Ras: the CAAX motif targets proteins to the ER and Golgi. Cell. 1999; 98(1):69-80; Wang G, and Deschenes R J. Plasma membrane localization of Ras requires class C vps proteins and functional mitochondria in Saccharomyces cerevisiae. Mol Cell Biol. 2006; 26(8):3243-55).
Since farnesylation is prerequisite for Ras biological activity (Hancock J F, Magee A I, Childs J E, and Marshall C J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989; 57(7):1167-77; Casey P J, Solski P A, Der C J and Buss J E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci USA. 1989; 86(21):8323-7; Rowinsky E K. Lately, it occurs to me what a long, strange trip it's been for the farnesyltransferase inhibitors. J Clin Oncol. 2006; 24(19):2981-4) farnesyltransferase inhibitors (FTIs) were thought to be excellent anti-Ras drugs. However, in cells treated with FTIs K- and N-ras are alternatively prenylated by geranylgeranyl transferase 1 (GGTase1) and traffic normally from the ER to the plasma membrane (Rowinsky, et al., 2006, ibid; Sebti S M, and Der C J. Opinion: Searching for the elusive targets of farnesyltransferase inhibitors. Nat Rev Cancer. 2003; 3(12):945-51). Geranylgeranylated oncogenic K- and N-ras are as transforming as the cognate farnesylated proteins (Hancock J F, Cadwallader K, Paterson H, and Marshall C J. A CAAX or a CAAL motif and a second signal are sufficient for plasma membrane targeting of ras proteins. EMBO J. 1991; 10(13):4033-9; Cox, A. D., Hisaka, M. M., Buss, J. E. & Der, C. J., Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein. Mol Cell Biol 1992; 12: 2606-15). Inhibition of the two post-prenylation enzymes Rce1 and Icmt has attracted considerable attention, as post-prenylation reactions are shared by both farnesylated and geranylgeranylated proteins. However, since Rce1 and Icmt act on more targets than the prenylation enzymes, toxicity is a concern with the post-prenylation inhibitors (Panagiotis A. Konstantinopoulos, Michalis V. Karamouzis1 & Athanasios G. Papavassiliou, Nature Reviews Drug Discovery 2007; 6: 541-555).
It has previously been shown that K-ras must be localized to the plasma membrane in order to activate downstream effector pathways (Hancock, J. F. Ras proteins: different signals from different locations, Nat Rev Mol Cell Biol 2003; 4: 373-84; Hancock, J. F. & Parton, R. G., Ras plasma membrane signaling platforms. Biochem J. 2005; 389: 1-11). Therefore inhibition or blocking of K-ras association with the plasma membrane would provide a much needed method of inhibiting K-ras signaling and turning off genes involved in erroneous cell growth and proliferation, and thus provide methods of treating oncogenic K-ras disorders. Thus, the present disclosure addresses such need, and provides methods that identify inhibitors of K-ras localization to the plasma membrane, and thus provide methods of inhibiting the signal transduction from oncogenic K-ras, which may be used as therapies for cancer, such as though not limited to, leukemias, colorectal cancers, pancreatic cancers and lung cancers.